Fix & Perm Kit, Ready-to-Use, IC-3016
Product Introduction
This product is a fixation and permeabilization kit containing ready-to-use cell fixation solution and cell permeabilization solution, designed for intracellular antigen staining in flow cytometry and immunofluorescence experiments. In immunological research, many critical targets such as cytokines, transcription factors, and phosphorylated signaling proteins are located in the cytoplasm or nucleus, where conventional surface staining cannot achieve detection. Cells must be fixed to maintain antigen morphology and localization, while permeabilization allows macromolecules such as antibodies to penetrate the cell membrane and bind to intracellular targets. This kit employs a mild fixation and permeabilization protocol that effectively fixes cells and selectively permeabilizes cell membranes while preserving cell surface and intracellular antigen immunoreactivity, cell morphology, and light scatter characteristics. It is suitable for intracellular staining analysis by flow cytometry and immunofluorescence microscopy. The kit is compatible with multiple fluorophore-conjugated antibodies and is widely used for immunophenotyping, intracellular cytokine detection, signaling pathway studies, and rare cell population analysis.
Product Features
1. Ready-to-Use: Both fixation and permeabilization solutions are ready-to-use working solutions requiring no dilution or preparation.
2. Mild Formulation: Mild fixation and permeabilization conditions balance intracellular antigen exposure with cell morphology preservation.
3. Compatibility: Preserves cell surface antigens and light scatter properties, suitable for surface and intracellular multi-color staining.
4. Convenient Protocol: Simple two-step procedure with standardized workflow.
5. Broad Application: Compatible with both flow cytometry and cell immunofluorescence staining platforms.
Specifications
Size: 50 tests / 100 tests
Components: Fixation Solution (ready-to-use), Permeabilization Solution (ready-to-use)
Product Type: Cell fixation and permeabilization kit
Applications: Flow cytometry, immunofluorescence staining
Storage and Stability
Storage Conditions: Store at -20°C for 1 year; store at 4°C for 1 month. Aliquot and store at -20°C is recommended. Avoid repeated freeze-thaw cycles. Thaw required amount and equilibrate to room temperature before use.
Shelf Life: 1 year at -20°C; 1 month at 4°C.
Protocol (For Reference Only)
1. Cell Preparation: Collect cell suspension, centrifuge at 300-400 x g for 5 minutes, discard supernatant. Wash once with appropriate volume of PBS, centrifuge and discard supernatant. Resuspend cell pellet in PBS to appropriate concentration (e.g., 1×10⁶ cells/100 µL). Record the volume of resuspension.
2. Surface Staining (Optional): If surface antigen detection is required, perform surface antibody staining at this step. Wash once with PBS, centrifuge and discard supernatant. Resuspend in PBS equal to the original cell suspension volume.
3. Fixation: Centrifuge cell suspension at 300-400 x g for 5 minutes, discard supernatant. Add Fixation Solution equal to the original cell suspension volume to the cell pellet. Gently vortex to mix. Incubate at room temperature for 15-20 minutes protected from light.
4. Washing: Add appropriate volume of PBS or PBS containing 1% BSA. Centrifuge at 300-400 x g for 5 minutes, discard supernatant. Repeat wash once if needed.
5. Permeabilization and Intracellular Staining: Add Permeabilization Solution equal to the original cell suspension volume to the cell pellet. Add fluorescent antibodies for intracellular antigens. Gently vortex to mix. Incubate at room temperature for 20-30 minutes protected from light.
6. Washing: Add appropriate volume of PBS or PBS containing 1% BSA. Centrifuge at 300-400 x g for 5 minutes, discard supernatant. Repeat wash once.
7. Analysis: Resuspend cells in appropriate volume of PBS or flow cytometry buffer. Analyze by flow cytometry as soon as possible.
Precautions
1. Both Fixation Solution and Permeabilization Solution are added to the cell pellet after centrifugation and supernatant removal. The volume added should equal the original cell suspension volume before centrifugation. Do not add directly to the cell suspension.
2. Fixation Solution and Permeabilization Solution have different formulations and are not interchangeable. Do not mix or use simultaneously.
3. Protect fluorophore-conjugated antibodies from light throughout the procedure to prevent photobleaching.
4. Fixation and permeabilization may affect certain antigen epitopes. Antibody titration optimization is recommended for first use.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: Should I add fixation/permeabilization solution directly to the cell suspension or to the pellet after centrifugation?
A1: Centrifuge the cell suspension first, discard supernatant, then add fixation or permeabilization solution to the cell pellet at a volume equal to the original cell suspension volume. Do not add directly to the cell suspension.
Q2: What should I do if cell number is significantly reduced after fixation and permeabilization?
A2: Cell loss during fixation and permeabilization is normal. Prepare slightly more cells than expected and minimize cell loss during repeated washing and supernatant aspiration.
Q3: What causes weak intracellular staining signal?
A3: Possible causes include insufficient permeabilization preventing antibody entry, over-fixation masking antigen epitopes, or inadequate antibody concentration. Extend permeabilization time, increase antibody concentration, or try an alternative antibody clone.
Q4: Cell morphology changes after fixation/permeabilization. How to set FSC/SSC gates?
A4: Changes in scatter characteristics (decreased FSC, increased SSC) are normal after fixation and permeabilization. Set initial gates based on unfixed cells, then adjust gating strategy using fixed/permeabilized negative control cells.
Q5: How should I aliquot and store the fixation and permeabilization solutions?
A5: Upon first use, aliquot fixation and permeabilization solutions into single-use volumes and store at -20°C protected from light. Thaw one aliquot per experiment. Avoid repeated freeze-thaw cycles of the entire bottle.
Q6: Can fixed cells be stored long-term?
A6: Fixed cells can be stored at 4°C protected from light for 24-48 hours, but long-term storage is not recommended. For longer storage, fix stained cells with 1% paraformaldehyde and store at 4°C protected from light. Analyze as soon as possible.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for use in food, drugs, or storage in ordinary residences.
2. Due to the exploratory, uncertain, and multi-variable nature of scientific research, a small-scale pilot experiment should be conducted to confirm product performance before application in large-scale critical experiments.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data. Purchase constitutes acceptance of these terms.
4. Wear appropriate protective clothing and disposable gloves when handling this product.
Ordering Information
Catalog Number: IC-3016
Product Name: Fix & Perm Kit, Ready-to-Use
Size: 50 tests / 100 tests
Suggested Retail Price:
50 tests: CNY ¥810.00 / USD $81.00 / EUR �97.20 / JPY ¥14580.00
100 tests: CNY ¥1450.00 / USD $145.00 / EUR �174.00 / JPY ¥26100.00
Note: The specific price is subject to the transaction price in the offline contract. This document is provided for technical reference only and does not constitute any form of sales advertisement or invitation to offer. For product details and purchase inquiries, please contact local sales representatives.
